Non-Alcohol Hand Sanitiser Gels with Mandelic Acid and Essential Oils

Antimicrobial hand gels have become extremely popular in recent years due to the COVID-19 pandemic. Frequent use of hand sanitising gel can lead to dryness and irritation of the skin. This work focuses on the preparation of antimicrobial acrylic acid (Carbomer)-based gels enhanced by non-traditional compounds—mandelic acid and essential oils—as a substitute for irritating ethanol. Physicochemical properties (pH and viscosity), stability and sensory attributes of the prepared gels were investigated. Antimicrobial activity against representative Gram-positive and Gram-negative bacteria and yeasts was determined. The prepared gels with mandelic acid and essential oil (cinnamon, clove, lemon, and thyme) proved to have antimicrobial activity and even better organoleptic properties than commercial ethanol-based antimicrobial gel. Further, results confirmed that the addition of mandelic acid had a desirable effect on gel properties (antimicrobial, consistency, stability). It has been shown that the essential oil/mandelic acid combination can be a dermatologically beneficial hand sanitiser compared to commercial products. Thus, the produced gels can be used as a natural alternative to alcohol-based daily hand hygiene sanitisers.


Introduction
The skin of a healthy person contains resident bacteria. Transient microbiota from the environment can cause an infection or even a severe health disease [1]. Hand hygiene, including hand washing and hand disinfection, is considered an important and effective measure against the transmission of microorganisms. During recent pandemics, hygienic hand disinfectants have received widespread attention, especially in gel form [2]. The gel is considered to be a cosmetic vehicle of many forms, which can serve as a carrier of active substances transported to the target site. The consistency and texture of the gel are modifiable and may be tailored to the specific application [3,4]. The gel easily turns into a liquid and is quickly absorbed into the skin, which reduces the possibility of run-off. This is accompanied by lower consumption and economic benefits [1].
Recently, the global market has provided a wide range of gel products with antimicrobial properties, especially alcohol-based gels. In international standards and guidelines, alcohols are recognised to be the most effective and efficient antimicrobials with a vast spectrum of antimicrobial activity [5]. The recommended ethanol/isopropanol/n-propanol concentration is usually in the range of 60-95% v/v, which is suggested to control the occurrence of dangerous microorganisms on the hands. The disinfecting effect of alcohols increases with molecular weight and chain length [6]. Their mechanism of antimicrobial action is based on the ability to denature proteins. All vegetative forms of Gram-positive

Antimicrobial Activity In Vitro
The antimicrobial activity was tested by the disk diffusion method either on paper disks in the case of mandelic acid and essential oils or in wells in agar in the case of prepared gels.

Mandelic Acid and Essential Oils
The disk diffusion method was used to determine the antimicrobial activity of substances that were subsequently used to prepare gels, namely mandelic acid (5, 10 and 15% solutions) and essential oil (EO-cinnamon CEO, clove CLEO, lemon LEO and thyme TEO). The results summarised in Table 1 show that all tested microorganisms were sensitive to all tested compounds except for lemon essential oil, which did not show any inhibitory activity against Gram-negative bacteria (E. coli and P. aeruginosa). On the other hand, the highest antimicrobial activity was observed by TEO, followed by CLEO and CEO. The antimicrobial effect of MA itself depended on its concentration. Already 10% of MA showed significant antimicrobial activity against all tested microorganisms. Table 1. Inhibitory activity of mandelic acid (MA) solutions and four essential oils (EOs) on selected microbial strains using the disk diffusion method (inhibition zones include 6 mm paper disk).
Gels containing Et (45 and 60%) were also prepared and tested to compare their antimicrobial effect with alcohol-free samples. GEt (45%) showed very low antifungal and antibacterial activity, whereas a higher Et concentration (60%) caused an increase in antimicrobial effect. GEO samples, except GLEO, exhibited comparable activity against P. aeruginosa and S. aureus and even better antimicrobial activity against M. luteus and yeasts than GEt 60%. The addition of MA significantly enhanced the antimicrobial activity, as was proved even by GCEO/MA samples against E. coli. Table 2. Inhibitory activity of gels containing essential oils (EOs) and/or mandelic acid (MA), or ethanol (Et) on selected microbial strains using the well diffusion method (inhibition zones include 6 mm well diameter).

Antimicrobial Activity In Vivo
The antimicrobial efficacy tests of prepared gels (GEO/MAs) in vivo were intended to simulate the practical conditions of hand sanitiser usage. The percent reduction in microorganisms ( Figure 1) presents the efficacy of the tested gels after antimicrobial hand gel application according to EN 1500.
The results reveal that the highest antimicrobial activity was observed with GCLEO/MA, followed by GCEO/MA and GTEO/MA. In comparison, GLEO/MA showed the lowest efficacy, nevertheless, similar to ethanol-based tested commercial samples. The hand microbiome is highly variable because it is affected by different surrounding factors. This study confirmed that prepared GEO/MAs reduced microbial counts of the artificially contaminated hands (except GLEO/MA) significantly better than ethanol-based commercial products. The pH was measured with time up to 60 days at three different temperatures (22 ± 1 • C, 37 ± 1 • C and 50 ± 1 • C). It was found that the temperature and storage time had no significant effect on the pH of all GEOs (Figures 2 and S1). The mandelic acid in GEO/MAs caused even more negligible decrease in pH during the time compared to GEOs. The results generally showed a slight drop in pH, regardless of temperature and storage time.

Effect of Temperature and Storage Time on pH and Viscosity
The pH was measured with time up to 60 days at three different temperatures (22 ± 1 °C, 37 ± 1 °C and 50 ± 1 °C). It was found that the temperature and storage time had no significant effect on the pH of all GEOs (Figures 2 and S1). The mandelic acid in GEO/MAs caused even more negligible decrease in pH during the time compared to GEOs. The results generally showed a slight drop in pH, regardless of temperature and storage time. As can be seen in Figures 2 and S1, the most significant reduction in viscosity was observed at the highest tested temperature, 50 °C, in all the samples due to the destruction of the gel structure network. At lower temperatures, the viscosity reduction was slower and more gradual, except in the GLEO gel, when viscosity dropped more than 50%.

Effect of Temperature and Storage Time on pH and Viscosity
The pH was measured with time up to 60 days at three different temperatures (22 ± 1 °C, 37 ± 1 °C and 50 ± 1 °C). It was found that the temperature and storage time had no significant effect on the pH of all GEOs (Figures 2 and S1). The mandelic acid in GEO/MAs caused even more negligible decrease in pH during the time compared to GEOs. The results generally showed a slight drop in pH, regardless of temperature and storage time. As can be seen in Figures 2 and S1, the most significant reduction in viscosity was observed at the highest tested temperature, 50 °C, in all the samples due to the destruction of the gel structure network. At lower temperatures, the viscosity reduction was slower and more gradual, except in the GLEO gel, when viscosity dropped more than 50%. As can be seen in Figure 2 and Figure S1, the most significant reduction in viscosity was observed at the highest tested temperature, 50 • C, in all the samples due to the destruction of the gel structure network. At lower temperatures, the viscosity reduction was slower and more gradual, except in the GLEO gel, when viscosity dropped more than 50%.
A significant decrease in the viscosity of GEOs occurred at 22 ± 1 • C within the first 10 days, after which the viscosity stabilised at approximately 6000 mPa.s. For gels containing TEO, CLEO and CEO, a slower and more gradual viscosity reduction was observed.
In the case of GEO/MA samples, no significant effect of MA addition on the viscosity was observed in GLEO samples during the entire testing period. On the other hand, a stabilising effect of mandelic acid on the gel structure can be confirmed in the samples with CLEO and CEO at almost all tested temperatures. Surprisingly, the TEO gel showed the opposite trend, with the most substantial drop (from approximately 17,000 to 2000 mPa.s) at 50 • C.
Comparison of the viscosity of the two series of samples, GEO and GEO/MA, showed that increasing temperature significantly reduced the viscosity of gels containing only EO. On the other hand, the addition of MA enhanced the gel stability and kept the viscosity high except GLEO/MA.

Effect of Temperature and Storage Time on Antimicrobial Efficacy and Organoleptic Properties
The antimicrobial activity of produced gels with mandelic acid (GEO/MA) against E. coli, S. aureus, and C. albicans was determined by the well diffusion method. Tests were performed immediately after the gel production (0 days) and after 28 and 60 days of storage at three temperatures (22 ± 1 • C, 37 ± 1 • C, and 50 ± 1 • C). The results for GCLEO/MA, GLEO/MA, and GTEO/MA are presented in Figure 3 (GCEO/MA) and Table S1 (Supplementary).
In the case of GEO/MA samples, no significant effect of MA addition on the viscosity was observed in GLEO samples during the entire testing period. On the other hand, a stabilising effect of mandelic acid on the gel structure can be confirmed in the samples with CLEO and CEO at almost all tested temperatures. Surprisingly, the TEO gel showed the opposite trend, with the most substantial drop (from approximately 17,000 to 2000 mPa.s) at 50 °C.
Comparison of the viscosity of the two series of samples, GEO and GEO/MA, showed that increasing temperature significantly reduced the viscosity of gels containing only EO. On the other hand, the addition of MA enhanced the gel stability and kept the viscosity high except GLEO/MA.

Effect of Temperature and Storage Time on Antimicrobial Efficacy and Organoleptic Properties
The antimicrobial activity of produced gels with mandelic acid (GEO/MA) against E. coli, S. aureus, and C. albicans was determined by the well diffusion method. Tests were performed immediately after the gel production (0 days) and after 28 and 60 days of storage at three temperatures (22 ± 1 °C, 37 ± 1 °C, and 50 ± 1 °C). The results for GCLEO/MA, GLEO/MA, and GTEO/MA are presented in Figure 3 (GCEO/MA) and Table S1 (Supplementary). All GEO/MAs stored at 22 ± 1 °C exhibited promising antimicrobial activity against all tested microorganisms even after 60 days of storage in comparison with higher tested All GEO/MAs stored at 22 ± 1 • C exhibited promising antimicrobial activity against all tested microorganisms even after 60 days of storage in comparison with higher tested temperatures. With increasing temperature, the antimicrobial efficacy decreased, especially against Gram-positive S. aureus. The decrease in effectivity can probably be explained by EO oxidation at a higher temperature. It was proved that the most significant effect GCEO/MA was against yeasts and Gram-positive bacteria. The comparable activity of GCEO/MA and GTEO/MA was noticed against Gram-negative bacteria. The lowest activity of all tested gels had GLEO/MA.
The visual and odour properties (colour and smell) of the produced gels (GEO and GEO/MA-15% solution) at defined time intervals were monitored ( Figure 4). These properties should maintain at an acceptable level throughout the time. All produced gels were milky cloudy with a corresponding typical smell of the respective EO after their preparation. This coloration was caused by the addition of EO. The smell of the produced gel changed along with the colour change over time and with increasing storage temperature.
tivity of all tested gels had GLEO/MA. The visual and odour properties (colour and smell) of the produced gels (GEO GEO/MA-15% solution) at defined time intervals were monitored ( Figure 4). These pr erties should maintain at an acceptable level throughout the time. All produced gels w milky cloudy with a corresponding typical smell of the respective EO after their prep tion. This coloration was caused by the addition of EO. The smell of the produced changed along with the colour change over time and with increasing storage temperat

Sensory Analysis
The organoleptic properties of the set of gel samples were assessed immediately a preparation with scales and subsequently evaluated with the Kruskal-Wallis test, wh uses rank sums in the calculation algorithm ( Table 3). The ranking sums correspond to interpretation of the assessed properties supplemented by testing the differences betw the gel samples at the 5% significance level. Sensory evaluation profiles were created fr the five obtained characteristics, see Figure 5. The appearance and colour were most often assessed as very good, correspondin products of the given type, transparent with a low presence of bubbles. Based on the s of the ranks, GLEO/MA sample was evaluated as the best, whereas ethanol-based co mercial gel SANYTOL received the highest score. A statistically significant differenc

Sensory Analysis
The organoleptic properties of the set of gel samples were assessed immediately after preparation with scales and subsequently evaluated with the Kruskal-Wallis test, which uses rank sums in the calculation algorithm ( Table 3). The ranking sums correspond to the interpretation of the assessed properties supplemented by testing the differences between the gel samples at the 5% significance level. Sensory evaluation profiles were created from the five obtained characteristics, see Figure 5. The appearance and colour were most often assessed as very good, corresponding to products of the given type, transparent with a low presence of bubbles. Based on the sum of the ranks, GLEO/MA sample was evaluated as the best, whereas ethanol-based commercial gel SANYTOL received the highest score. A statistically significant difference in this characteristic was not found. As regards the fragrance parameter, GLEO/MA was again the best-rated sample, while GTEO/MA was given the highest number of points. The calculation revealed conclusive statistical differences, specifically between the samples GCEO/MA-GTEO/MA, GLEO/MA-SANYTOL and GLEO/MA-GTEO/MA. GTEO/MA with the typical gel texture received the lowest sum, while commercial SANYTOL gel was graded with the highest. There was a statistically significant difference between the SANYTOL-GTEO/MA gel samples. The results of spreadability are consistent with these texture founding, and GTEO/MA was identified as the best spreadable. From the set of samples, the commercial ethanol-based gel was marked with the least satisfactory spreadability. There was a statistically significant difference in this monitored property between the GCEO/MA-SANYTOL, GCLEO/MA-SANYTOL and GTEO/MA-SANYTOL samples. Absorbency was the last monitored characteristic using scales. The commercial SANYTOL gel showed very good and fast absorbency. On the other hand, GCEO/MA received the highest score, with poor absorbency resulting in a sticky film on the skin. However, no statistically significant difference in absorbency was found among the samples. this characteristic was not found. As regards the fragrance parameter, GLEO/MA was again the best-rated sample, while GTEO/MA was given the highest number of points. The calculation revealed conclusive statistical differences, specifically between the samples GCEO/MA-GTEO/MA, GLEO/MA-SANYTOL and GLEO/MA-GTEO/MA. GTEO/MA with the typical gel texture received the lowest sum, while commercial SANYTOL gel was graded with the highest. There was a statistically significant difference between the SANYTOL-GTEO/MA gel samples. The results of spreadability are consistent with these texture founding, and GTEO/MA was identified as the best spreadable. From the set of samples, the commercial ethanol-based gel was marked with the least satisfactory spreadability. There was a statistically significant difference in this monitored property between the GCEO/MA-SANYTOL, GCLEO/MA-SANYTOL and GTEO/MA-SANYTOL samples. Absorbency was the last monitored characteristic using scales. The commercial SANYTOL gel showed very good and fast absorbency. On the other hand, GCEO/MA received the highest score, with poor absorbency resulting in a sticky film on the skin. However, no statistically significant difference in absorbency was found among the samples.
The evaluation of the sequential preference test proved that the evaluated samples did not differ in preferences. Based on the sum of the rankings, see Table 3, the assessors preferred GLEO/MA, followed by GCEO/MA, GCLEO/MA, gel with TEO/MA, and the commercial gel SANYTOL was marked last in the ranking.

Discussion
This work aimed to prepare hand sanitiser with alternative non-alcohol antimicrobial agents to avoid the adverse skin effect of ethanol. As Houben et al. [22] examined, it was The evaluation of the sequential preference test proved that the evaluated samples did not differ in preferences. Based on the sum of the rankings, see Table 3, the assessors preferred GLEO/MA, followed by GCEO/MA, GCLEO/MA, gel with TEO/MA, and the commercial gel SANYTOL was marked last in the ranking.

Discussion
This work aimed to prepare hand sanitiser with alternative non-alcohol antimicrobial agents to avoid the adverse skin effect of ethanol. As Houben et al. [22] examined, it was found that a higher concentration of ethanol resulted in increased skin scaling during tests with six alcohol-based antimicrobial gels.
At the initial stage, the antimicrobial properties of the used EOs, MA and their combination were tested. The results showed that the antimicrobial activity of essential oils is mainly affected by their specific composition, functional groups present in active components, and their synergistic interactions [23]. Therefore, their antimicrobial efficacy in produced gels can be different as well. For example, the antimicrobial effects of CEO can be attributed to its main component, cinnamaldehyde, which can disrupt the membranes of bacteria, leading to cell lysis [24]. Goñi et al. also investigated the antibacterial activity of CEO in combination with CLEO against Escherichia coli, Yersinia enterocolitica, Pseudomonas aeruginosa, Salmonella choleraesuis, Staphylococcus aureus, Listeria monocytogenes, Enterococcus and Bacillus [25]. The main components responsible for the antimicrobial activity of TEO include carvacrol, thymol, p-cymene, È-terpinene [16]. Cinnamaldehyde, eugenol, limonene and thymol, as main components of essential oils used in this study, have been shown to have also antiviral activities [26]. Other characteristics, such as solubility in water and ability of hydrogen bonding play important role in final antimicrobial activity, too. It has been already reported in the literature that although thymol, carvacrol and eugenol exhibit strong antimicrobial effects, these can be eliminated by high water solubility. This can be the reason of lower activity obtained in CLEO samples [27]. Thymol and carvacrol, as two components with highly similar structure, have been proved to have additive interaction [28]. It was found that the combination of linalool and eugenol exhibited a synergistic effect [18].
The antimicrobial activity of EOs was also confirmed in other works [29,30]. It is generally known that Gram-positive bacteria are more sensitive to the action of EOs than Gram-negative bacteria, mainly because of the cell wall structure [31,32]. According to Nazzaro et al. [32], the antibacterial effect of EOs and their components are associated with their lipophilic nature, which allows them to accumulate in membranes and thus act on cell wall destruction. Some studies [33,34] reported that selected EO could replace conventional antimicrobials. The addition of MA enhanced the antimicrobial effects of prepared gels. Similarly, Stickler et al. proved the in vitro antimicrobial properties of MA and their derived compounds. The effectiveness of MA (1% w/v) and MA (0.5% w/v)/lactic acid (0.5% v/v) mixture in eliminating the biofilm forming organisms related to urinary tract infections was shown [35].
The hand microbiome was found to be a more temporal variable than in other body sites. It consists of bacteria, fungi, viruses, and protozoa. Bacteria from four phyla were observed-Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes. The hand microbiome is affected by temporal and biogeographical dynamics, as well as intrinsic and extrinsic factors [36]. While Staphylococcus aureus is a common cause of skin infection, methicillinresistant S. aureus (MRSA) represents a severe cause of morbidity and mortality in many hospitals worldwide. The hands of health care workers are the most common vehicle for transmitting pathogens, so hand hygiene is very important in preventing and controlling MRSA [37]. MA derived compounds were tested for their antimicrobial activity against a few Gram-positive and Gram-negative bacteria by the disk diffusion method. They demonstrated the antimicrobial effect of MA at concentrations of 20-160 mg/mL against MRSA [19]. Thus, mandelic acid could be an effective treatment for MRSA and other bacterial skin infections.
Evidence from the literature search shows that several antimicrobial gels have been recently investigated [38][39][40]. Liu et al. [39] described the fabrication of PEG hydrogels that incorporate an antimicrobial polycarbonate (polycarbonate containing quaternary ammonium groups), forming cationic PEG-APC (polyethyleneglycol-antimicrobial polycarbonate) hydrogels. These gels showed strong antimicrobial activity against S. aureus, E. coli, and C. albicans. In addition, the prepared hydrogel proved to maintain its activity over time even when challenged daily with S. aureus for 12 days.
The results from the present study revealed that the antimicrobial activity of GEO and GEO/MA samples was significantly affected by the temperature and total storage time. The findings correspond to the work of Turek et al. [41], which states that rising temperature alter the stability of EOs and their chemical composition during storage. Those changes can negatively influence the resulting antimicrobial efficacy.
The viscosity of the gels plays a crucial role in their functionality, as the high viscosity facilitates contact time. Hydrogel preparations can be more desirable than liquid forms, especially from the viewpoint of user comfort. They absorb faster, have a shorter drying time, and leave a pleasant feeling on the hands. In addition, they can reduce the rate of alcohol evaporation [42]. Regarding the organoleptic and rheological properties of gel formulations, they are generally influenced by the used thickeners, active substances, and their concentrations [43]. In topical formulations, they may be perceived differently by the end-user when applied to the skin, not only during but also after application. The sensory influence of the ingredients was described, for example, in the publication [44], which investigated skin protection hydrophilic gels based on polyacrylates. The acrylic derivates have proven as good thickeners creating hydrophilic films on the surface of the skin. Hydroxyethyl cellulose and Carbomer of synthetic nature in the studied gels were used as gelling agents in the study of Savary et al. [45], who confirmed the good absorption properties of prepared samples. Similarly, the gels prepared in this study exhibited optimal absorption properties when no residual film was observed after their application on the skin. On the other hand, the tested commercial gel was evaluated as poorly spreadable, which can be the result of its thin texture. This indicates that the developed antimicrobial formulations outperformed the commercial formulation, especially in this subjectively assessed parameter.
Diffusion methods were used for four bacterial and two yeast strains. Bacterial strains Escherichia coli CCM 3954, Staphylococcus aureus CCM 3953, Pseudomonas aeruginosa CCM 3955, Micrococcus luteus CCM 734, and yeast strains Candida albicans CCM 8275, and Candida parapsilosis CCM 8276 were supplied by Czech Collection of Microorganisms (Brno, Czech Republic). Bacteria were cultivated on Nutrient agar or Mueller Hinton agar; yeasts were grown on Sabouraud agar (Hi-Media Laboratories Ltd., Mumbai, India).

Preparation of Gels
Four types of gels were prepared: GEt (with ethanol), GEO (with essential oils), GMA (with mandelic acid), and GEO/MA (with essential oils and mandelic acid). The formulations of prepared gels are shown in Table 4. The gel base was Carbomer (0.5 wt%), which was weighed into a 250 mL beaker, and demineralised water was supplemented (100 g). The swollen Carbomer gel base and ingredients (Et or EO) were thoroughly mixed for 10 min on a stirrer (MM4 LAVAT, CZ). Subsequently, GEt, and GEO gels were adjusted to pH 6 with 1 M NaOH solution.
For the preparation of gels enriched with mandelic acid (GMA), MA solutions of different concentrations (5, 10, 15%) were initially prepared. Then, the pH was adjusted to approx. 3.5 using 1 M NaOH solution. This acidified MA solution was added in the appropriate amount (5.0 wt%) to the swollen Carbomer gel base. In the case of GEO/MA gels, the addition of acidified MA solution was followed by the incorporation of the corresponding essential oil (0.5 wt%). The mixture was stirred vigorously for 10 min (MM4 LAVAT, CZ), and the pH of this solution was adjusted to pH 5.6-6.0 with 1 M NaOH solution.

Viscosity Measurement
The viscosity of the produced gels was measured at laboratory temperature (22 ± 1 • C) on a rotational viscometer MYR V2-L (Viscotech Hispania, SL, Spain) using the Brook-field method and evaluated using ViscosoftPlus software according to ISO 2555 [46]. All experiments were measured in triplicate.

pH Measurement
A pH meter (pH Spear Waterproof, USA) with a measurement accuracy of ± 0.1 pH was used to measure the pH of the produced gels. All measurements were taken at least three times for each value.

Antimicrobial Activity In Vitro
As the first step, the antibacterial activity of essential oils, mandelic acid and ethanol (control) was tested by the standard agar disk diffusion technique [47]. The sterile 6 mm disks (Whatman, Maidstone, UK) were impregnated with 10 µL active substance. They were placed on Mueller Hinton agar plates (Himedia Laboratories Pvt. Ltd., Mumbai, India) inoculated with 0.5 McF turbid bacterial suspension (M. luteus, S. aureus, E. coli and P. aeruginosa) in sterile saline solution and on Sabouraud agar (Himedia Laboratories Pvt. Ltd., Mumbai, India) inoculated with 0.5 McF turbid yeast suspension (Candida albicans, C. parapsilosis). The plates were incubated for 24 h at 37 • C for bacteria and 5 days at 25 • C for yeasts. The inhibition zones were measured in diameter, including the paper disk. All experiments were repeated three times.
The antimicrobial activity of prepared gels was tested by the well diffusion method, as was described earlier [48]. At first, 1 mL of each microbial suspension (0.5 McF) was pipetted into separate sterile Petri dishes and poured with 20 mL molten Mueller-Hinton agar (bacteria) or Sabouraud agar (yeasts). After solidification, wells of 5 mm diameter were made in the center of each agar plate by sterile cork borer, and 50 µL of gel was placed into it. The diameters of inhibition zones were measured. All experiments were performed at least in triplicate.

Antimicrobial Activity In Vivo
The efficacy of hygienic handrubs (GEO/MA) was tested by measuring the number of viable bacteria remaining on the fingertips of 18 volunteers according to EN 1500 [9]. All accepted volunteers had healthy, intact skin and they provided informed consent. Ethical approval was sought, but this was not required as the method used was based upon published EN standards, as well as institutional review board approval was not required in case volunteers involved in sensory analysis. Nevertheless, the selection of volunteers and the testing procedure were in accordance with international ethical principles of biomedical research with human participants [49]. This standard is designed to evaluate the ability of routine hand hygiene products to eliminate transient pathogens. This procedure involves the placement of bacteria on the hands followed by exposure to the test gel sanitiser [50]. The nonpathogenic bacterial strain Escherichia coli CCM 3954 was used in this study. Participants were asked to wash and dry their hands. The following step included artificial contamination of hand fingertips in bacterial suspension without (prevalue A) and with (post-value B) the hygienic handrub. Four GEO/MA with cinnamon, clove, lemon and thyme essential oil were tested. Two commercial ethanol-based hand sanitiser gels were used as comparative controls (BALEA 45% ethanol, SANYTOL 60% ethanol). Colony counts were obtained and log reductions were calculated. The logarithmic reduction factor was then expressed as a percent reduction. Log reduction was calculated according to Equation (1) [51]: where A is a Log number of viable microorganisms without treatment (pre-value); B is a Log number of viable microorganisms with treatment (post-value).

Sensory Analysis
Sensory analysis was performed by a panel of 12 sensory assessors with prior sensory evaluation experience (ages 21-26 years). They were familiarised with the process and purpose of sensory evaluation through questionnaires. The sensory evaluation and equipment of the sensory laboratory of Tomas Bata University, Faculty of Technology, met the defined conditions according to the international standards ISO 6658 [53] and ISO 8589 [54]. The laboratory temperature ranged from 20 to 22 • C, and the room was illuminated by artificial lighting. Sensory evaluation of gels was performed after signing informed consent before accepting participation in the study by all sensory assessors.
The sample formulations were assessed by scales and a ranking preference test. Properties such as appearance and colour were evaluated using an ordinal hedonic five-level scale (1-excellent, typical of the type of formulation, transparent, presence of bubbles, 2very good, appearance still adequate, transparent, moderate presence of bubbles, 3-good, transparent or slightly cloudy, no bubbles, 4-less good, appearance different, slightly yellowish, cloudy, no bubbles, 5-unsatisfactory, non-transparent, markedly cloudy and discoloured, no bubbles); smell (1-excellent, pleasant smell, 2-very good, no disturbing odours, 3-good, slightly disturbed by odours, 4-less good, much disturbed by odours, unsatisfactory-strong presence of foreign odours); texture (1-excellent, adequate gel texture, homogeneous, 2-very good gel texture with the presence of visible droplets of the oil phase, 3-good texture, slightly thin, with slight separation of the oil phase, 4-less good texture, thin with noticeable separation of the oil phase, 5-unsatisfactory texture, very thin, inhomogeneous with separating phases), spreadability (1-excellent typical for the product type, 2-very spreadable, slightly liquid, 3-well spreadable, moderately liquid, 4-poorly spreadable, liquid, 5-almost not spreadable, very liquid), absorbency (1-excellent and rapid absorption, leaves no sticky character, 2-very good absorption, no sticky film formation, 3-good absorption, leaves a slightly sticky impression, 4-poorer absorption with sticky film formation, 5-not absorbed, leaves a strongly sticky film). Spreadability and absorbency were evaluated during and after application of gels in the amount of 0.1 mL on an area of 8 cm 2 of the volar side of the left forearm.

Statistical Analysis
Data from antimicrobial activity tests (disk and well diffusion method) were expressed as the mean ± standard deviation (SD). Statistical analysis was carried out by a one-way ANOVA followed by a Tukey test using Statistica software version 10, StatSoft, Inc. (Tulsa, OK, USA) at the significance level of p < 0.05.
The Kruskal-Wallis test was used to compare the sensory trait using scales. Ordinal tests were evaluated by Friedman's test. The results of the sensory evaluation were processed at the 5% significance level (p < 0.05) using Unistat 5.5 software (Unistat Ltd., London, UK).

Conclusions
Alternative formulations for the commonly used antimicrobial component (ethanol) in gels intended for hand disinfection were investigated. The antimicrobial effect of the developed formulations was tested against selected physiologically significant Grampositive and Gram-negative bacteria and yeasts.
The synergistic action of MA and selected EOs in the gel formulations significantly supported the high inhibitory effects in both in vitro and in vivo experiments against tested microorganisms and on hands, respectively. The prepared GEO/MA gel showed higher antimicrobial activity than the commercial ethanol-based samples. The stability test results confirmed the significant effect of temperature on gels containing only EO, while the addition of MA had rather a supporting and stabilising effect in the gel samples regarding their viscosity, unfortunately not with regard to antimicrobial efficacy. Sensory analysis has revealed that the organoleptic properties of GCEO/MA, GCLEO/MA and GLEO/MA were better than the commercial ethanol-based product.
It can be concluded that the cinnamon essential oil combined with mandelic acid provided an enhanced antimicrobial activity in comparison to formulations based only on EO, especially against yeasts. This combination of active compounds could serve as a natural alternative to the alcohol component. The prepared gel formulations containing essential oils (0.5 wt% EO) and mandelic acid (10-15% MA solution) can be applied as daily hand skin hygiene, mainly due to their extended efficacy and biocompatibility.